BR112020023607A2 - systems and methods for focusing particles on microchannels - Google Patents

Abstract

The present invention relates to a microfluidic system configured to focus particles suspended in a fluid. A general aspect includes a microfluidic system comprising one or more substrates and a focusing channel formed on one or more substrates and covering a length from an inlet to an outlet to receive a flow of particles suspended in fluid, the particles have a diameter (a ) and the focusing channel has a hydraulic diameter (dh).

Description

Descriptive Report of the Invention Patent for “SYSTEMS AND METHODS FOR FOCUSING PARTICLES ON MICROCANALS”.

BACKGROUND OF THE INVENTION

[001] Flow cytometers work by passing individual particles, like cells, into a fluid stream through a detector, which measures certain characteristics of each particle and performs actions based on this assessment. To do this, the flow cytometer must regulate the sample flow so that the particles in the sample move into a substantially single-row particle flow, which allows each particle to be measured individually by the detector.

[002] One area in which flow cytometers have found practical use is the connection with sexing of sperm, such as bovine sperm, according to the characteristics of the sperm cells for use by the animal breeding industry to pre-select the sex of the offspring animal. The most common method for sexing sperm cells is to discriminate based on the content of DNA. In this context, the sperm is combined with an extender and a luminescent dye to stain the DNA within the sperm cell. The spotted sperm cells are then placed in a sample fluid that is introduced into a channel of a microfluidic chip that uses focusing techniques to orient the sperm cell in a substantially single-row stream. After being properly oriented, the sperm are illuminated with a light source (for example, a laser), which excites the luminescent dye in the DNA, emitting a fluorescent luminescence that is discovered by a detector (for example, a photomultiplier tube (" PMT ”) or an avalanche photodiode (APD)). A sperm containing the X chromosome has more DNA than a sperm carrying the Y chromosome, resulting in the sperm carrying the X chromosome producing more luminescence in response to the light source of The detected luminescence is monitored and the system takes a selective action, for example, classifying or killing unselected sexed sperm with a deadly laser, on the individual sperm cells to achieve a final product with the desired characteristics, for example, a sample with a high concentration of sperm with X or Y chromosomes. For example, if calves are desired (for example, for the production of le ite), then the system is calibrated to collect cells having detected luminescence parameters that are what would be expected of a sperm cell carrying the X chromosome. Alternatively, if calves are desired (for example, for meat production), then the system is calibrated to collect cells having detected luminescence parameters that are what would be expected of a sperm cell carrying the Y chromosome.

[003] Sperm cells can also be differentiated based on DNA content by other methods that do not use a DNA dye. For example, US Patent No. 8,941,062 describes cytometric systems and methods involving the presentation of a single sperm cell to at least one laser source configured to provide light to the sperm cell in order to induce binding vibrations in DNA of the sperm cell and detect the signature the vibrations of the connection. Sperm cells can also be analyzed and differentiated based on the presence or absence of cell surface markers or protein, by binding a fluorescently labeled ligand, such as an antibody. Other methods for discriminating sperm cells can use other characteristics of sperm cells, such as mass or volume, to differentiate between those containing X chromosomes and those containing Y chromosomes. These discrimination and detection methods allow cells to be selectively differentiated and that the sample is sexed.

[004] Sexing techniques include a variety of methods to classify, separate, eliminate or inactivate unwanted cells. For example, so-called laser destruction methods involve exposing specific cells to a laser with enough energy to inactivate the cells. Cells can also be separated into populations by sorting, for example, through droplet formation and deflection, as described in U.S. Patent No. 5,700,692.

[005] In cell discrimination techniques, including sperm cell sexing applications, the orientation, ordering and proper location of cells within the microfluidic system are essential for effective operation. For example, positioning and orientation are both essential to being able to effectively detect the difference in fluorescence of sperm cells with X and Y chromosomes stained with an intercalating DNA dye, as well as the positioning of cells within the laser beam of Detection and orientation of cells in relation to the detector has a significant impact on the amount of fluorescence detected. Changes in fluorescence, in turn, directly affect the ability to distinguish differences in the fluorescence signal between cells carrying the X chromosome and the Y chromosome. In addition, during the sexing process, the various techniques used depend on the ability to locate accurately the cells within the fluid stream. For example, in laser destruction sexing, the destruction laser is closely focused on a specific point and requires cells to be positioned properly in order for the exposure to be effective in inactivating the cell. The positioning of cells within the flow (that is, up, down, left and right, relative to the axis of displacement) and the ordering (that is, the distance between cells along the axis of displacement) also they are important for classification techniques (ie formation and deflection of droplets, classification of thermal bubbles, etc.). The ordering of cells in a sample stream can be non-deterministic (that is, it follows a Poisson distribution) or deterministic (that is, spacing). The ordering, therefore, refers to the control of the incidence of cells in the sample flow.

[006] Hydrodynamic focusing has been used to align cells, including sperm cells, in flow cytometry applications for many years, but it can have disadvantages. First, hydrodynamic focusing can involve multiple streams of fluid, including one or more streams of fluid acting in a shell. The casing fluid is consumable in the flow cytometry process and can be costly. In addition, hydrodynamic targeting can involve microfluidic chips being designed and produced with complex samples and sheath flow channels, leading to relatively high costs for microfluidic chips. The number of flow controllers required for each number of fluid inlets can also impact the cost. In addition, hydrodynamic focusing depends on a consistent flow rate of the enclosure flow and fluctuations in the flow rate, for example, due to a loss of pressure or occlusion of an enclosure channel, can have adverse effects on the performance of the cytometer .

[007] Inertial flow targeting has been used for cell types, such as white blood cells and cancer cells. These cells are significantly larger than sperm cells and are uniform in shape (that is, substantially spherical). In contrast, sperm are significantly smaller, non-uniform and have tails. As a result, the balance of forces acts differently in sperm cells than in other types of cells.

SUMMARY OF THE INVENTION

[008] Certain embodiments of the claimed invention are summarized below. These embodiments are not intended to limit the scope of the claimed invention, but rather to serve as brief descriptions of possible forms of the invention. The invention can cover a variety of forms that differ from these summaries.

[009] According to one embodiment, particle focusing is achieved using a microchannel, where the ratio of the particle diameter (a) to the hydraulic diameter of the channel (Dh), defined by the formula a / Dh, is between approximately 0.03 and about 0.06, and / or where the curvature ratio (radius "r" or "critical parameter") to the hydraulic diameter of the channel, defined by the formula 2ra2 / Dh3, is less than approximately 0, 03.

[010] As used in this document, "focusing" refers to the spatial organization of cells in a desired formation, in particular, in a defined spatial width with reference to an axis along which the cells are moving in a microfluidic channel and / or in relation to a defined reference point, such as the detection focus point or the destruction laser or both). In one embodiment, a focused flow of cells will all be 3-5 times within a given cell dimension (i.e., width, height or length) of the center line of the displacement axis. In other embodiments, a focused flow of cells will be within 2-3 times the size of the cell and in other embodiments within 1-2 times the size of the cell.

[011] Upon entering the microfluidic system, cells are initially defocused (that is, not within the desired spatial parameters); various forces can act on the cells within the flow stream to bring them within the desired spatial parameters (that is, the cells are focused).

[012] Other aspects will be evident to a person skilled in the art after reviewing the description and exemplary aspects and embodiments that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[013] For the purpose of illustrating the disclosure, certain characteristics of the aspects and embodiments of the disclosure are represented in the drawings. However, the disclosure is not limited to the precise arrangements and instrumentalities of the aspects represented in the drawings.

[014] Figure 1A shows a repeatedly curved part of a microchannel according to certain aspects of an embodiment of the present disclosure. Figure 1B shows an inertial targeting project modified according to certain aspects of an embodiment of the present disclosure. Figure 1C shows a modified modified inertial targeting project according to certain aspects of an embodiment of the present disclosure. Figure 1D shows an inertial targeting project according to certain aspects of an embodiment of the present disclosure.

[015] Figures 2A and 2B show enlarged views of a curved segment of an embodiment of the microchannel of Figure 1.

[016] Figures 3A and 3B show enlarged views of a curved segment of another embodiment of the microchannel of Figure 1.

[017] Figures 4A and 4B show the critical parameter or radius of curvature "r".

DETAILED DESCRIPTION

[018] Before proceeding to describe various aspects and embodiments in more detail, it should be understood that this disclosure is not limited to specific compositions or process steps and may vary. As used in this specification and the appended claims, the singular form "a", "an" and "o" includes plural referents, unless the context clearly dictates otherwise. The ranges expressed here are inclusive.

[019] An embodiment of the microfluidic chip can be used in connection with sexing sperm cells, such as bovine sperm, for example. In particular, the chip can be used in a device that uses flow cytometry to sex sperm cells according to DNA characteristics for use by the animal breeding industry to pre-select the sex of the animal offspring. Soon, the sperm is combined with an extender and a luminescent dye to stain the DNA inside the sperm cell. The dye-stained sperm cells are then placed in a sample fluid that is introduced into a microfluidic chip channel. Since sperm cells are not spherical, the microfluidic chip substantially guides sperm cells to reduce differences in luminescence detection that can be caused by differences in cell orientation relative to the detector.

[020] The targeted sperm cells are then illuminated with a light source (for example, detection laser), which excites the luminescent dye in the DNA, emitting a fluorescent luminescence that is detected by a detector (for example, a photomultiplier tube ( PMT) or an avalanche photodiode (APD)). The sperm containing the X chromosome has more DNA than the sperm carrying the Y chromosome, resulting in the sperm carrying the X chromosome producing more luminescence in response to the original illumination. The difference in the total DNA content varies by species; for example, in Bos taurus, the X chromosome has approximately 3.8% more DNA than the Y chromosome, which results in an approximately 3.8% difference in fluorescence.

[021] In order to determine which cells to kill, an output signal from the detector that represents the amplitude of the detected luminescence is monitored. When the detected luminescence value exceeds a defined limit value, an event is considered to have started. The luminescence value is monitored and, when an inflection point or “peak” is detected, the peak is considered the center of the cell and the peak luminescence value is considered the luminescence value for that cell. If more than one peak is detected in a single event, the peak with the highest amplitude is considered the center of the cell and the peak luminescence value is considered the luminescence value for that cell and the other peaks are disregarded.

[022] The luminescence value of each sperm is compared to a port, which has been previously defined, to determine whether the cell exhibits the desired luminescence. For example, if calves are desired (for example, for milk production), then the port is selected to include cells having detected luminescence parameters that are what would be expected of a sperm cell carrying the X chromosome. Alternatively , if calves are desired (for example, for meat production), then the port is selected to include cells having detected luminescence parameters that are what would be expected of a sperm cell carrying the Y chromosome.

[023] After passing through the detection laser and having its luminescence detected, the stained sperm, still in the flow, then move on to the destruction zone. A second light source, for example, the destruction laser, is selectively activated to kill cells that fall outside the selected door as they pass through the destruction zone.

[024] In other embodiments, the particle concentration according to the present invention can be used for different sperm cells based on the DNA content by methods that do not use a DNA dye. For example, US Patent No.

8,941,062 describes cytometric systems and methods involving the presentation of a single sperm cell to at least one laser source configured to provide light to the sperm cell in order to induce binding vibrations in the sperm cell's DNA and detect signature the vibrations of the connection. In other embodiments, sperm cells can be analyzed and differentiated based on the presence or absence of cell surface markers or protein, by binding a fluorescently labeled ligand, such as an antibody. Other methods for discriminating sperm cells can use other characteristics of sperm cells, such as mass or volume, to differentiate between those containing X chromosomes and those containing Y chromosomes. These discrimination and detection methods allow cells to be selectively differentiated and that the sample is sexed. In other embodiments, sperm cells can be differentiated based on characteristics other than sex. For example, sperm cells can be differentiated based on the presence or absence of a genetic marker or combination of markers or cell surface protein.

[025] In other embodiments, particle focusing, as described in this document, can be used for semen sexing techniques to classify, separate, eliminate or inactivate unwanted cells. For example, so-called laser destruction methods involve exposing specific cells to a laser with enough energy to inactivate the cells. Cells can also be separated into populations by sorting, for example, through droplet formation and deflection, as described in U.S. Patent No. 5,700,692. Other classification techniques for use in the present invention include, for example, bubble classification, acoustics, photonic pressure, holographic laser targeting and optical capture.

[026] The microfluidic chip according to the present project uses a microchannel curved repeatedly to order and focus particles in the sample fluid mixture. The chip can be composed of one or more substrates on which the channel, or part of the channel, is formed. The substrate can be composed of one or more layers. The channel is a three-dimensional structure within one or more layers assembled from one or more substrates. In one embodiment, the chip may include two layers, a lower layer and an upper layer, which are stacked together to form the chip. In one embodiment, a repeatedly curved part of the microchannel is formed entirely in the lower layer, while the inlets and outlets for the microchannel can be formed in one or both layers of the chip. In other embodiments, the microchannel can be formed in two or more layers of one substrate, or multiple substrates. The curved part repeatedly consists of a repeated series of identical shaped loops, as shown in Figure 1.

[027] In use, a sample fluid is introduced into the microchannel through a sample inlet. In the context of bovine semen, the sample includes an ejaculate and a buffer. Upon entering the microchannel, the particles are randomly dispersed within the fluid sample. As the sample flows, the microchannel of the particles is ordered longitudinally so that, when leaving the curved part, the particles are aligned longitudinally in a row. The microchannel can include horizontal / lateral and / or vertical tapering downstream of the curved portion to provide additional focusing of the particles before the fluid moves through a detection region (not shown).

[028] Figures 2A and 2B are enlarged views of a curved segment of an embodiment of the microchannel of Figure 1. Figures 3A and 3B are enlarged views of another embodiment of the microchannel of Figure 1.

[029] The channels described above, which allow only a single focusing position, due to Dean's flow regulation effect, comprise 1.5 turns (Figure 2A and Figure 3A) or a single turn (Figure 2B and Figure 3B) . With reference to Figures 2B and 3B, each loop includes a smaller region; the critical parameter is indicated (0.015 mm). In both Figures 2 and 3, each loop also includes a larger region (that is, the bottom of the loop). The smaller region and the larger region are staggered. Together, a smaller region and a larger region constitute a single loop of the microfluidic channel. The curves shown in Figures 2 and 3 are asymmetric, in which the smaller and larger regions are different, and the general curve, therefore, is not internally symmetrical. In other embodiments, the curve can be symmetrical, in which the left and right sides (i.e., the upper and lower parts of the curve) have the same geometries. In other embodiments, the loops within a focusing channel can include one or more symmetrical loops, one or more asymmetric loops or combinations thereof. In other embodiments, the focusing channels can include still other hydraulic mechanisms for effecting the positioning, orientation and / or ordering of particles within the sample stream.

[030] Figures 4A and 4B illustrate how to measure the critical parameter "r".

[031] As shown in Figure 4A, the cross-sectional area is determined by the height (H) and width (W) of the microchannel in the smallest region of a turn. Figure 4B shows the radius determination of the smallest region of a curve.

[032] According to one aspect, particle focusing is achieved using a microchannel curved repeatedly:

[033] Where the ratio of the particle diameter (a) to the hydraulic diameter of the channel (Dh), defined by the formula a / Dh is between approximately 0.03 and approximately 0.06, and / or where the ratio of curvature (radius "r" or "critical parameter") for the hydraulic diameter of the channel) defined by the formula 2ra2 / Dh3 is less than approximately 0.03.

[034] In another aspect, the particle may be bovine sperm cells. Bovine sperm cells are irregularly shaped and sperm cells are smaller, non-uniform (~ 3 pm thick x 5 pm wide x 10 pm long) and have a tail (picometer (pm): 10−12 meters). In this context, the cell diameter is considered to be in the range of 3 pm to approximately 5 pm. The targeting of substantial bovine sperm particles is observed when the geometries of the microfluidic channels meet one or both of the above conditions. However, if the physical geometries fall outside these ranges, for example, if the / Dh is greater than approximately 0.06 or less than approximately 0.03, the bovine sperm cells do not concentrate.

[035] Any number of microfluidic system configurations can be designed to achieve certain results and / or specific properties associated with the concentration of particles within the various channel geometries. In the examples below, certain properties associated with the systems described in this document will now be discussed in more detail. Although certain experimental conditions can be discussed with reference to certain properties or parameters, it should be understood that the properties and parameters are widely applicable to any of the channel geometries. Example 1

[036] In one aspect, the inertial focusing design (Figure 1D) was tested on polydimethylsiloxane (PDMS) and glass at 4 different flow rates. The measurements were taken from the width of the central flow, flat percentage and edge percentage and are shown in Table 1. The numbers are shown for PDMS, and where the measurements differ when made on glass chips are shown in parentheses.

[037] The width of the core flow (eg, W_68, W_95, W_100) is the measured width of a certain percentage of the core flow, as measured using images taken by a sample strobe flowing through the microfluidic chip. For example, W_68 is the measured width of 68% of the main flow.

[038] The flat percentage, which is measured on a strobe, is the measurement of cells that are oriented with the widest cross section parallel to the top of the channel and therefore also perpendicular to the detector and the ablation laser beam paths .

[039] The edge percentage, which is measured on a strobe, is the measure of cells with the narrowest cross section perpendicular to the top of the channel and therefore parallel to the laser beam paths.

Table 1 68% wide 95% wide 100% wide Flow rate% Flat% Core edge of core core 37- 50% 23 - 29% 250 µL / min 6-8 µm 12 - 16 µm 24 - 30 µm (53 - 68%) (7-13%) 22 - 27% 300 µL / min 4.5 - 7.5 µm 9- 14 µm 13-33 µm 42-50% (58-67%) 5 - 7% 15 - 21% 45-62% (52- 350 µL / min 5-7 µm 10 - 12.5 µm 14-28 µm 68%) 6 - 9% 12- 20 µm 15 - 31 µm 22 - 24% 400 µL / min 5 - 10 µm 42-47% (43-68%) (10-12 µm) (18 - 21µm) 7- 8% Example 2

[040] In one aspect, the modified inertial focusing design (Figure 1C) was tested. The modified design incorporated an upstream element that includes a curvature in the channel in combination with the dilution on the chip to obtain the focus of the sample in the Z dimension (that is, from top to bottom, in relation to the direction of travel). The parameters for the curvature of this upstream element were 200pm in radius and 100pm in width (R200W100), 300pm in radius and 100pm in width (R300W75) and 500pm in radius and 100pm in width (R500W100). The dilution was tested with a dilution of 5%, 10%, 12.5% and 20%. The inertial focusing part of the modified design is identical to the inertial focusing design tested in Example 1 and the measurements reported in Table 2 include core current width, flat percentage and edge percentage. Table 2 68% wide 95% wide 100% wide Edge% Core dimensions% Plan Core core 5/10 / 12.5 / 20% of curvature dilution

5/10 / 12., 5/20% 5/10 / 12.5 / 20% 5/10 / 12.5 / 20% 5/10 / 12.5 / 20 dilution (live.) Dilution dilution% of dilution 13.3 / 14.1 / 14.0 / 13, 6µm R200W10 7.7 / 8.5 / 7.9 / 8.1µm 53/53/51/47% 11/16/15/19% 3, 9 / 4.3 / 3.9 / 40.0 µm (4.0). 0 (8.0) (51%) (15%) (13.8) - - / 3.6 / 3.8 / 4.0µm - / 7.1 / 7.5 / 8 - / 58/54 / 52% - / 10/9/7% / 10.1 / 12.4 / 14.1µm R300W75 (3.8) (7.5) (54%) (8%) (12.2) 4.2 / 3.5 / 6.3 / 4.8µ R500W10 8.4 / 7.0 / 12.5 / 9.5µm 18.3 / 7.8 / 16.4 / 12.0 65/57/58/62% 11/8/11/13% m 0 (9.4) (13.6) (60%) (11%) (4.7) Example 3

[041] In another aspect, the different modified design was tested, which incorporated a downstream element that includes a curvature in the channel without any dilution on the chip (Figure 1B). In another aspect, the secondary region downstream of the inertial focusing channel in this project included a secondary input to potentially adjust the entry point of the cells in the curved section of the drift element, although this element was not used during the test. The data in Table 3 provide the same type of results obtained for the modified and unmodified inertial targeting projects discussed in the previous examples. Measurements were made using a flow rate of 300pl / min. Table 3 68% wide 95% wide 100% wide% Edge% core core core Flat 8 µm 16µm 20 µm (50%) (11%) Although illustrative embodiments have been illustrated and described, it will be appreciated that several changes can be made to them, without departing from the spirit and scope of the invention.

Claims (5)

1. Microfluidic system to focus particles suspended in a fluid, characterized by the fact that it comprises: one or more substrates; a focusing channel formed on one or more substrates and spanning a length from an inlet to an outlet to receive a flow of particles suspended in fluid, the particles have a diameter (a) and the focusing channel has a hydraulic diameter (Dh) , in which: a. wherein the ratio of the particle diameter to the cross-sectional area of the channel (a / Dh) is between approximately 0.03 and approximately 0.06, and / or b. where the curvature ratio (critical parameter; "r") to the hydrodynamic curvature (Dh) defined by the formula 2ra2 / Dh3 is less than approximately 0.03. 2. Microfluidic system, according to claim 1, characterized by the fact that the microchannel comprises a repeatedly curved segment. Microfluidic system according to claim 2, characterized by the fact that the curved segment repeatedly comprises a series of repetition of curved sections of identical shape. 4. Microfluidic system according to claim 3, characterized by the fact that the curved sections of identical shape are asymmetrical. Microfluidic system according to any one of claims 1 to 4, characterized in that the particles comprise bovine sperm.